an apoptosis enhancing drug overcomes platinum resistance in a tumour initiating subpopulation of ovarian cancer

While treatment with carboplatin enriches for CA125-negative cells, co-treatment with carboplatin and birinapant eliminates these cells in HGSCs expressing high levels of the inhibitor o

An apoptosis-enhancing drug overcomes platinum resistance in a tumour-initiating subpopulation

of ovarian cancer

High-grade serous ovarian cancers (HGSCs) are deadly malignancies that relapse despite

carboplatin chemotherapy Here we show that 16 independent primary HGSC samples

contain a CA125-negative population enriched for carboplatin-resistant cancer initiating cells.

Transcriptome analysis reveals upregulation of homologous recombination DNA repair and

anti-apoptotic signals in this population While treatment with carboplatin enriches for

CA125-negative cells, co-treatment with carboplatin and birinapant eliminates these cells in

HGSCs expressing high levels of the inhibitor of apoptosis protein cIAP in the CA125-negative

population Birinapant sensitizes CA125-negative cells to carboplatin by mediating

degradation of cIAP causing cleavage of caspase 8 and restoration of apoptosis This

co-therapy significantly improves disease-free survival in vivo compared with either therapy

alone in tumour-bearing mice These findings suggest that therapeutic strategies that target

CA125-negative cells may be useful in the treatment of HGSC.

1Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA

2Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA.3Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA.4The VA Greater Los Angeles Health Care System, Los Angeles, California 90073, USA Correspondence and requests for materials should be addressed to S.M

(email: smemarzadeh@mednet.ucla.edu)

T he efficacy of high-grade serous ovarian cancer (HGSC)

treatment has not improved significantly since the advent

of platinum-based chemotherapy1, with 5 year survival

at 30–40% in advanced stage disease despite radical surgery

and chemotherapy1 Following first-line treatment disease is

undetectable in predominance of patients, yet most relapse

within 6–16 months2 Relapsed patients are treated with

repeated chemotherapy, but over time response to carboplatin

diminishes.

Despite global efforts, imaging coupled with measurement of

the biomarker CA125 has proven ineffective in early detection of

serous ovarian cancers3 On a therapeutic front, efforts have

focused on supplementing platinum drugs with agents that target

specific genetic defects4,5 or strategies that can reverse the

platinum-resistant phenotype6 Mechanisms proposed for

platinum resistance in HGSCs include accumulation of genetic

mutations, epigenetic alternations, and influences from the

microenvironment7,8 The leading hypothesis in the field

assumes that many HGSCs are innately platinum sensitive but

with chemotherapy exposure platinum refractory clones emerge9.

Over time, tumours shift to a platinum-resistant phenotype as

these cells come to take over the cancer through clonal evolution.

Mutations that correlate with platinum resistance have been

documented in some cases of serous cancer8,10, but this alone

may not explain the almost universal resurgence of HGSC after

first-line treatment with platinum drugs An alternative model

that could explain high rates of relapse dominated by a platinum

refractory phenotype is innate platinum resistance in subsets of

tumour cells with cancer initiating properties present in all

HGSCs Regrowth of these therapy-resistant cells could result in

relapse of disease despite platinum chemotherapy and aggressive

surgical measures8.

Previous work suggests that HGSC contain a tumour-initiating

population of cells but a universal marker for their isolation has

not been identified11–14 This could be due to the use of cell lines

and xenografts with unstable cancer initiating populations13and

the application of stem cell markers from other malignancies to

characteristics of stem cells found in their tissue of origin16 As

mounting evidence suggests HGSC may originate from the

fallopian tube17–19, we defined fallopian tube epithelial

progenitors and discovered these cells were CA125 negative17.

Here we demonstrate that subsets of cells in human HGSCs are

CA125 negative and possess stem characteristics of tumour

initiation, multi-lineage differentiation and self-renewal While

treatment with carboplatin eliminates differentiated

CA125-positive HGSC cells, the CA125-negative population

is innately platinum resistant Upregulation of inhibitor of

apoptosis proteins (cIAP) is one mechanism enabling evasion

of platinum-induced cell death in CA125-negative HGSC cells.

Pharmacologic targeting of cIAP with birinapant in HGSCs with

high cIAP levels in their CA125-negative population sensitizes

these therapy-resistant cells to platinum resulting in their

elimination and a significant increase in disease-free survival.

Findings here pave the way for understanding why HGSCs

commonly recur despite platinum treatment We demonstrate

that addition of birinapant to carboplatin chemotherapy can

eliminate HGSC cells in subsets of tumours by mechanistically

re-enabling apoptosis in the CA125-negative population.

Results

CA125-negative HGSC cells have cancer initiating capacity.

CA125 (Muc16), a cell surface glycoprotein20highly expressed in

HGSC and shed into the bloodstream20, is a commonly used

serous cancer biomarker While majority of HGSC cells express

CA125, we hypothesized the cancer initiating cells would be CA125 negative as fallopian tube epithelial progenitors do not express CA125 (ref 17) and CA125 is ineffective in early detection of HGSC21.

To test this hypothesis, CA125 expression was examined by fluorescent-activated cell sorting (FACS) in 16 chemo-naive primary HGSC patient specimens (Supplementary Table 1, Fig 1a, Supplementary Data 1 Supplementary Fig 1a,b and Supplementary Data 2) In all samples a clear CA125-negative HGSC population was detected (19.3±9.8% median±interquartile range (IQR), n ¼ 16; Fig 1a, Supplementary Data 1 and Supplementary Fig 1c) CA125-negative and -positive sub-populations contained a mixture of cells expressing epithelial (EpCAM) and/or stromal (CD10) markers, and both fractions contained cells expressing CD44 (Supplementary Fig 1d,e and Supplementary Data 2) CA125-negative populations were significantly higher in HGSCs not amenable to complete resection (sub-optimal cytoreduction) compared with tumours that could

be totally removed on initial surgery (optimal cytoreduction) (29.4±10.0% (n ¼ 6) versus 16.8±6.7% (n ¼ 10), median±IQR;

P ¼ 0.004 unpaired two-sided t-test; Fig 1a and Supplementary Data 1) As poorer survival outcomes are achieved when patients have sub-optimal cytoreduction, higher percentages of CA125-negative cells may be associated with more aggressive HGSCs22 In addition, analysis of The Cancer Genome Atlas23 database revealed a trend towards decreased overall survival with lower CA125 mRNA levels (Supplementary Fig 1f and Supplementary Data 2).

The in vivo growth of CA125 subpopulations from chemo-naive human HGSC was compared using fresh or live-banked cryopreserved cells, shown to have similar cancer initiating capacities (Supplementary Fig 2) Equal numbers of matched CA125 negative, CA125 positive or bulk primary HGSC cells (105 cells in each inoculum) were grown as subcutaneous xenografts for 6 months (Fig 1b) In 10 of 10 solid tumours and 2 of 4 ascites samples, CA125 negative but not CA125-positive HGSC cells generated a tumour (Fig 1c) In two ascites samples, larger tumours were produced by the CA125-negative subpopulation while small tumours were generated from CA125-positive cells (Fig 1c, patients 11 and 12) In all cases, significantly larger xenografts were generated from CA125-negative cells than from matched bulk HGSC cells (n ¼ 14, Po0.01 repeated measure analysis of variance (ANOVA); Fig 1d and Supplementary Data 1).

High-grade serous cancer stem cells are CA125 negative Previous work demonstrates that HGSCs follow a cancer stem cell model13, but the identity of the cancer stem cells remains elusive11–14 Our results demonstrate that CA125-negative HGSC cells were enriched for tumour-initiating capacity (Fig 1c,d and Supplementary Data 1), suggesting this population may contain HGSC stem cells.

To test this hypothesis, limiting dilution, lineage tracing and passaging assays for stem activity were used24 The tumour-initiating capacities of HGSC subpopulations were compared through in vivo limiting dilution using seven independent chemo-naive human HGSCs Two months after implantation of each tumour subpopulation in serial logarithmic dilutions, cancer initiating capacity was scored based on the presence of histologically confirmed tumour (Fig 2a, Supplementary Data 1 and Supplementary Fig 3a) In line with previous reports13, 1/42,000 (median, n ¼ 7) bulk tumour cells initiated a cancer (Supplementary Table 2) CA125-negative HGSC cells had a 670-fold greater tumour-initiating frequency than matched CA125 positive counterparts (1/1,100 versus 1/740,000 cells;

median, n ¼ 7 per group; Supplementary Table 2), and in five of

the seven HGSCs 100–1,000 CA125-negative cells initiated

tumours (Fig 2a, Supplementary Data 1 and Supplementary

Fig 3a).

As cancer stem cells possess the ability to differentiate25, a

CA125-negative population containing HGSC stem cells should

give rise to CA125-positive and -negative progeny To test this

hypothesis, in vivo lineage tracing was performed using the two

ascites samples that generated tumours from both CA125

subpopulations (Fig 1c patients 11 and 12) Equal numbers of

CA125-negative and -positive cells, permanently color-marked

using a GFP-expressing lentivirus, were injected in vivo Unlike

CA125-negative fractions, which generated tumours containing

GFP-marked CA125-negative and -positive cells recapitulating

the population distribution in the parent tumour, GFP-marked

progeny predominantly expressed CA125 in the small tumours

generated from CA125-positive cells (Fig 2b and Supplementary

Fig 3b) Findings suggest that while CA125-negative cells are

capable of multi-lineage differentiation, the CA125-positive

population is lineage restricted with limited growth potential.

To compare self-renewal of CA125 subpopulations, equal

numbers of unsorted cells from the ascites xenografts (Fig 1c

patients 11 and 12) were passaged in logarithmic dilutions

(Fig 2c) While xenografts derived from CA125-negative

populations contained both CA125-positive and -negative cells

and grew robustly, the other xenografts were composed primarily

of CA125-positive cells and had limited growth with passaging (Fig 2c and Supplementary Fig 3c) These results suggest that self-renewing HGSC cells are located predominantly in the CA125-negative subpopulation A recent report suggests that CA125-positive cells may also have tumour-initiating capacity26 However this report has a number of shortcomings which include (a) lack of controls for cell isolation and tumour take, (b) absence

of stringent assays for stem activity and (c) supplementation of tumour-bearing mice with exogenous oestrogen creating a hormonal milieu not physiologic in epithelial ovarian cancer patients.

Findings here demonstrate that the CA125-negative sub-population of human HGSC contains cancer stem cells that can initiate tumours, differentiate and self-renew Most patients with HGSC relapse despite having normal CA125 levels after therapy Relapse of tumour from residual CA125-negative cells, undetected by CA125 based assays, may occur in these patients This last notion implies that the CA125-negative tumour cells may be resistant to existing therapies.

Carboplatin-resistant HGSC cells are CA125 negative As cancer stem cells efficiently re-seed disease8, an inherently platinum-resistant tumour-initiating population could explain recurrence rates for HGSC Studies have demonstrated

0 10 20 30 40

50 P = 0.004

Optimally cytoreduced chemo-naive

HGSC (n =10)

Sub-optimally cytoreduced chemo-naive

HGSC (n =6)

Fraction CA125-negative cells in chemo-naive human HGSC

Solid tumors

Pt 1

Solid tumor

&

matched ascites

Pt 2

Pt 3

Ascites

Pt 11

Pt 9 (T)

Pt 10 (T)

Pt 9 (A)

Pt 4

CA125+

tumor cells

Bulk tumor cells

CA125 – tumor cells

1,000

10

0

Pt 7

Pt 5

Pt 2

Pt 8

Pt 3

Pt 1

Pt 4

Pt 6

Pt 9

Pt 9A

Pt 10

Pt 10A

Pt 11A

Pt 12A

P <0.01 P <0.01

100

Primary human

HGSC

100,000 CA125 + cells

100,000 CA125 – cells

CA125

Dissociated cells

from fresh (n =5)

or cryopreserved

(n =7) human HGSC

Pt 5

Pt 6

Pt 7

Pt 8

Pt 12

Pt 10 (A)

Figure 1 | A minor population of serous tumour cells that do not express CA125 are capable of in vivo growth (a) Aggressive tumours not amenable to

initiation assay Equal numbers of CA125-negative and -positive cells FACS isolated from fresh (n¼ 5) or live-banked (n ¼ 7) HGSCs were injected in vivo and grown for 6 months (c) The CA125-negative cells were the only population capable of generating tumours in eight solid and two matched solid tumour and ascites specimens In two ascites samples, tumour outgrowth was noted from both populations; however larger tumours were generated from CA125-negative cells (d) The weight distribution of xenografts arising from CA125-negative, CA125-positive and bulk tumour cells are shown for all

chemo-resistant behaviour in subsets of serous cancer cells27–34,

but much of this work has utilized cell lines that may not

recapitulate HGSC biology35or non-serous tumour models27–34.

We hypothesized that carboplatin would fail to kill

CA125-negative cells; implying tumours should be enriched for these cells

following therapy When comparing tumours from

chemo-treated (n ¼ 7) to chemo-naive (n ¼ 16) patients, the percentage

of CA125-negative cells increased twofold (median 46.2% (n ¼ 7)

versus 19.3% (n ¼ 16), P ¼ 0.003 unpaired two-sided t-test; Fig 3a

and Supplementary Data 1) To determine if this reflected an

increased pool of chemo-resistant tumour cells, the cancer

initiating capacities of HGSC cells obtained from the same

patient before and after carboplatin therapy were compared (Supplementary Fig 4a, n ¼ 3 independent specimens) Larger tumours developed from cells isolated post-chemotherapy (Supplementary Fig 4b, n ¼ 3), suggesting platinum treatment enriched for cancer initiating cells To rapidly test the efficacy of therapies in targeting HGSC subpopulations, an in vitro screen was established (Supplementary Fig 5 and Supplementary Data 2) When chemo-naive HGSCs were treated with carboplatin in this assay, differentiated tumour cells died but disproportionate numbers of CA125-negative cells survived (Fig 3b and Supplementary Data 1, n ¼ 9) Collectively, these results suggest that the CA125-negative cells are platinum resistant.

2/2 3/3 1/3 0/3 Tumor take

frequency hHGSC 1

0/2 0/3 0/3 0/3 1/2 0/3 0/3

CA125–

# Cells injected

Tumor take frequency hHGSC2

0/3

1 10 100 1,000 10,000 100,000 1,000,000 10,000,000

Bulk cells CA125–

cells CA125+

cells

Pt 4

Pt 2

Pt 3

Pt 1

Pt 6

Pt 7

Pt 5

Frequency of tumor-initiating cells based on extreme limiting dilution analysis

0/3

CA125+

3/3 3/3 3/3 3/3 2/3 0/3 0/3 0/3 Tumor take frequency

Equal logarithmic dilutions of dissociated unsorted cells from each xenograft

Tumor initiated from CA125 – CA125+

3/3 3/3 3/3 2/3

# Cells injected tumor take frequency

0/3 0/3

1/3

CA125–

Ascites sample 1

Ascites sample 2

TIC = 1/91 (CA125–) TIC = 1/280,000 (CA125+)

TIC = 1/21 (CA125–) TIC = 1/107,000 (CA125+)

CA125+ graft CA125 – graft

CA125+

95.1%

CA125 – 2.5%

CA125+

80.7%

CA125 – 16.1%

GFP+

57.8%

GFP+

60.4%

Lin-depleted CA125 – graft

Lin-depleted CA125+ graft

Lineage Lineage

Post-sort analysis CA125 – fraction

Infect with GFP-virus and implant

Infect with GFP-virus and implant

Lineage-depleted primary hHGSC

10.2% 83.3%

CA125

Post-sort analysis CA125+ fraction

Figure 2 | The CA125-negative population from primary human high-grade serous tumours contains the serous cancer stem cells (a) In vivo limiting dilution analysis demonstrates that CA125-negative cells have a significantly greater tumour-initiating capacity compared with matched CA125-positive

independent clinical specimens are shown The frequency of tumour-initiating cells for seven independent HGSCs is graphed Results are median±IQR Scale bars equal 5 mm (b) To enable lineage tracing of tumour subpopulations primary HGSCs were sorted based on CA125 expression, infected with a GFP-expressing lentivirus and grown in vivo Flow cytometry and immunohistochemistry demonstrated the presence of GFP-marked CA125-positive and -negative cells in the tumour generated from the CA125-negative subpopulation Conversely, most color-marked cells in tumours generated from the CA125-positive subpopulation were CA125 positive Scale bars equal 5 mm for gross xenografts of 50 mm for hemotoxylin and eosin stains and immunostains (c) Compared with the cells from CA125-negative xenografts, limited capacity for self-renewal was observed on in vivo passage of cells from

Carboplatin causes DNA damage that induces apoptosis36.

Mechanisms implicated in platinum resistance include: (a) drug

efflux/inactivation, (b) evasion of apoptosis, (c) DNA repair and

(d) resistance to autophagocytosis10,37 To explore platinum

resistance mechanisms exploited by the CA125-negative

population, the transcriptomes of CA125-negative and -positive

tumour cells from 10 independent chemo-naive human HGSCs

were compared (Fig 3c and Supplementary Data 3) This analysis

confirmed shared TP53 mutations in CA125-positive and -negative cells from the same tumour (Supplementary Table 3) Higher levels of transcripts involved in DNA repair and evasion

of apoptosis, but not drug efflux or autophagy, were found in the CA125-negative cells (Fig 3d,e, Supplementary Data 1 and Supplementary Tables 4 and 5) Elevated levels of the homologous recombination protein Rad51 and its loading partner E2F1 (Fig 3d), and differences in anti-apoptotic

CA125 – tumor cells

CA125+

tumor

10 CA125-positive tumor cell libraries

10 hHGSC specimens

10 CA125-negative tumor cell libraries

RNA sequencing for comparison

of transcriptome

CA125-positive differentiated cells CA125

P = 0.003

0 10 20 30 40 50 60

Chemo-naive HGSC

(n =16)

Chemotherapy treated HGSC

(n =7)

Fold transcript increase CA125– compared with

DNA repair

0 2.0 4.0

RAD51 BRCA2 RPA ATR

–4.0 –2.0 0 2.0 4.0

FAS FADD DR5 BAD Noxa APAF1

Fold transcript increase CA125– compared with

Apoptosis

CA125-positive differentiated tumor cells CA125-negative cells

E2F1

Rad51

Erk

+ cont – CA125 – CA125 +

Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6

Fas

Fadd

Erk

86.8%

84.2% cell death 2.5% cell death

–80 –70 –60 –50 –40 –30 –20 –10

hHGSC

CA125 – CA125 + CA125 – CA125 + CA125 – CA125 + CA125 – CA125 + CA125 – CA125 +

+ cont – cont. CA125 – CA125 +

Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6

CA125 – CA125 + CA125 – CA125 + CA125 – CA125 + CA125 – CA125 + CA125 – CA125 +

38 kDa

38 kDa

38 kDa

50 kDa

50 kDa

28 kDa

38 kDa

80 kDa

50 kDa

98.7%

1.1%

13.4% CA125

SSC SSC

FACS-isolated CA125-negative and CA125-positive tumor cell-populations

pan-cIAP

Figure 3 | The CA125-negative serous cancer cell population is resistant to platinum-based therapies (a) Administration of chemotherapy led to a disproportionate loss of differentiated CA125-positive cells versus CA125-negative cells when comparing tumours from chemo-naive (n¼ 16, compiled data from Fig 1a) to chemo-treated (n¼ 7) patients (results are median±IQR, P ¼ 0.003 unpaired two-sided t-test) (b) In vitro treatment of primary HGSC cells with carboplatin resulted in elimination of the differentiated cells but survival of the CA125-negative HGSC subpopulation Representative FACS plot is shown for one clinical sample, and fold-decrease in surviving cells is shown for nine independent clinical samples (mean±s.d., n¼ 3 replicates per sample) Scale bar equals 50 mm (c) Comparison of the transcriptomes from therapy-resistant CA125 negative and differentiated CA125-positive cell fractions of 10 independent primary human chemo-naive HGSCs revealed clear differences between the two populations Scale bar equals 5 mm (d) Higher transcript and protein levels for homologous recombination DNA repair genes were detected in therapy-resistant CA125-negative cells Results are mean fold change±s.e.m., n¼ 10 (e) The CA125-negative subpopulation had higher levels of anti-apoptotic and lower levels of pro-apoptotic transcript and protein levels Results are mean fold change±s.e.m., n¼ 10

(Fig 3e, cIAP1 and cIAP2) and pro-apoptotic protein expression

(Fig 3e, Fas and Fadd) were detected in CA125-negative

compared with -positive populations.

Birinapant sensitizes CA125-negative cells to carboplatin.

Platinum-induced DNA damage triggers apoptosis through

release of cytochrome c, second mitochondrial activator of

caspase (SMAC) and TNFa (refs 38,39) cIAP proteins inhibit

apoptosis by (a) binding to SMAC and preventing its interaction

with X-linked-inhibitor of apoptosis (XIAP) and (b) preventing

TNFa mediated activation of caspase 8 while simultaneously

enabling pro-survival signalling through NF-kB38,40–42 As

CA125-negative cells in subsets of HGSC harboured high levels

of cIAP (Fig 3e), we hypothesized that pharmacologic targeting

of cIAP with the SMAC mimetic birinapant would sensitize these cells to carboplatin by re-enabling apoptosis We chose to test birinapant in these assays as (a) it is already in clinical trials (NCT01681368 and NCT01940172) and (b) it has a 50-fold greater affinity for cIAPs than XIAP43.

Examination of carboplatin response in HGSC subpopulations revealed that though the CA125-negative cells sustain double strand DNA breaks similar to CA125-positive differentiated cells, they may repair the damage more quickly (Fig 4a and Supplementary Data 1, n ¼ 3) When birinapant was added

to carboplatin and tested in vitro against nine independent chemo-naive human HGSCs, bulk tumours cells were eradicated

CA125 positive differentiated HGSC cells

γH2Ax γH2Ax γH2Ax γH2Ax

foci over time CA125-negative therapy resistant HGSC cells

0 20 40 60 80 100 120

P = 0.02 P < 0.0001

Time after carboplatin administration

CA125 + differentiated tumor cells

CA125 – therapy resistant tumor cells

Highly sensitive to

birinapant (n = 6)

(patients 1– 4,7,9)

0 20 40 60 80 100 120

V

Less sensitive to

birinapant (n = 3)

(patients 5,6,8)

V

+ cont – cont

pan-cIAP

Erk

Pt 7 Pt 8 Pt 9

CA125+ CA125

In vitro growth of previously treated

then passaged cells bulk HGSC populations

0 20 40 60 80 100 120

Birinapant sensitive

HGSC (n = 6)

Birinapant less sensitive

HGSCs (n = 3)

B+C: Birinapant + carboplatin

20 40 60 80 100 120

V B C B+C

CA125 positive cells

Bulk tumor cells

0

Survival of primary chemo-naive hHGSC subpopulations after treatment (n=3 primary HGSC)

CA125 negative cells

38 kDa

50 kDa

B: Birinapant B+C: Birinapant + carboplatin

B+C: Birinapant + carboplatin

γH2Ax γH2Ax

γH2Ax γH2Ax

C: Carboplatin

V: Vehicle C: Carboplatin C: Carboplatin

Figure 4 | Birinapant co-treatment sensitized CA125-negative primary HGSC cells to carboplatin (a) DNA damage, detected by gH2Ax immunostain, was examined in CA125-negative and -positive populations from three independent primary chemo-naive human HGSCs at 12, 24 and 36 h

and Po0.0001 at 36 h, unpaired two-sided t-test) Results are mean±s.e.m., n ¼ 3 HGSCs 5 fields of view each Scale bars equal 100 mm (b) Birinapant and carboplatin co-administration resulted in eradication of all tumour cells only in clinical samples with high cIAP levels in the CA125-negative population (results are mean±s.e.m., n¼ 6 patients 1–4, 7 and 9 versus n ¼ 3 patients 5–6 and 8, sample run in triplicate) Cell survival was FACS quantitated based

on propidium iodide and AnnexinV negativity Western blot of pan-cIAP levels in CA125-negative and -positive subpopulations for patients 7–9 is shown (c) Following in vitro treatment with birinapant and carboplatin co-therapy or monotherapy with either agent, primary chemo-naive human HGSC cells were re-plated in fresh media No regrowth was observed from birinapant-sensitive HGSC (blue) following co-therapy In contrast, growth was noted in all specimens after treatment with birinapant or carboplatin as a single agent, or after co-therapy in specimens with low levels of cIAP in their CA125-negative population (pink) Results are mean±s.e.m (n¼ 6 birinapant sensitive and n ¼ 3 birinapant less sensitive, samples run in triplicate) (d) CA125-negative,

birinapant or both drugs Compared with vehicle-treated cells, carboplatin alone effectively eliminated the differentiated cells but did not kill the CA125-negative cells On average birinapant as a single agent was equally effective against both the CA125-negative and -positive cells (P¼ 0.085 unpaired two-sided t-test) but did not cause complete cell death in either population Results are mean±s.e.m., n¼ 3 HGSC run in triplicate

in the six specimens with high cIAP expression in their

CA125-negative population (Fig 4b and Supplementary Data 1

(n ¼ 6, patients 1–4 in Fig 3e and patients 7 and 9 in Fig 4b)) In

contrast, this therapy was ineffective in eliminating many tumour

cells in the three specimens with lower expression of cIAP in their

CA125-negative population (Fig 4b and Supplementary Data 1

(n ¼ 3, patients 5–6 in Fig 3e and patient 8 in Fig 4b)) The

response was durable based on in vitro passaging (Fig 4c and

Supplementary Data 1, n ¼ 6 birinapant sensitive versus n ¼ 3

birinapant less sensitive) To determine if this drug treatment

specifically targeted the CA125-negative cells, response to

therapy was tested in CA125 negative versus CA125-positive

subpopulations of HGSC (Fig 4d and Supplementary Data 1,

n ¼ 3 independent birinapant-sensitive specimens) Though

carboplatin monotherapy readily induced apoptosis and death

in differentiated cells, apoptotic cell death was observed in the

CA125-negative population only after birinapant and carboplatin

co-therapy based on propidium iodide and AnnexinV expression

and poly (ADP-ribose) polymerase (PARP) cleavage (Fig 5 a,b,

n ¼ 3) Birinapant-induced degradation of cIAP but not XIAP

was observed in all populations tested (Fig 5b) and this activity

was TNFa dependent (Fig 5c and Supplementary Data 1, n ¼ 3).

As cIAP proteins are important mediators of pro-survival

signalling38,40, activation of NF-kB was examined in tumour

subpopulations Constitutive activation of NF-kB was detected in

both populations, which was decreased by birinapant

monotherapy and further diminished with carboplatin

co-therapy (Fig 5b) Diminution of NF-kB pro-survival signalling

by birinapant may have played a role in sensitizing the

CA125-negative cells to carboplatin But, we think re-enabling apoptosis

is the main mechanism by which this co-treatment eliminated

these therapy-resistant cells as despite constitutive NF-kB

activation (Fig 5b) and its pharmacologic blockade with

N4-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine (QNZ)44

(Fig 5c and Supplementary Data 1, n ¼ 3) in both populations,

carboplatin as a single agent could kill only the differentiated but

not CA125-negative cells.

Findings here demonstrate that birinapant can sensitize subsets

of HGSC to carboplatin, and response correlates with high levels

of cIAP in the CA125-negative subpopulation The mechanism

accounting for the efficacy of this treatment is through

re-enabling apoptosis in this therapy-resistant population.

In vivo co-therapy increased HGSC progression-free survival.

To facilitate in vivo testing of birinapant and carboplatin

co-therapy with sufficient power and further elucidate the

birinapant mechanism of action, three low passage cell lines

were developed from birinapant-sensitive chemo-naive primary

HGSCs: S1-GODL (TP53 mutant/BRCA WT), S3-GODL (TP53

null/BRCA WT) and S5-GODL (TP53 null/BRCA2 mutant) Cell

lines resemble their parent HGSCs based on CA125 distribution

and cancer initiating potential, DNA profile and transcriptome

analysis (Supplementary Fig 6a–c) These cell lines were chosen

as they contain different TP53 mutations and BRCA status.

Similar to primary HGSCs, in vitro therapy of these cells lines

with carboplatin eliminated CA125-positive but not -negative

cells, while addition of birinapant to carboplatin eliminated

all tumour subpopulations (Supplementary Fig 7a and

Supplementary Data 2, n ¼ 3 replicates) This therapeutic effect is

likely cIAP specific as knockdown of cIAP1 and cIAP2 together

was sufficient to sensitize all CA125-negative cells to carboplatin

even in the absence of birinapant (Supplementary Fig 7b and

Supplementary Data 2, n ¼ 3 replicates) Findings here suggest

that degradation of both cIAP1 and cIAP2 by birinapant may

be the main mechanism by which birinapant sensitizes the

CA125-negative HGSC to carboplatin therapy.

The efficacy of carboplatin and birinapant co-therapy was evaluated in a subcutaneous xenograft model, enabling comparison of HGSC cells to control cell lines In cohort 1, mice carried xenografts from S1-GODL, Skov3 (birinapant sensitive, carboplatin insensitive45,46), Ovcar-3 (carboplatin sensitive, birinapant insensitive7,46) and MCF7 (birinapant and carboplatin insensitive45) cells injected as single inoculums into separate sites of the same mouse Mice in cohort 2 harboured tumours from S3-GODL, Skov3, S5-GODL and Ovcar-3 cells injected in a similar manner (Supplementary Fig 8a) Tumour establishment was confirmed before treatment with vehicle, birinapant, carboplatin or both drugs (n ¼ 8 per group, Fig 6a) Xenografts were analysed for treatment efficacy at the completion

of therapy and for disease progression 4 weeks after cessation of treatment (Fig 6a) To evaluate therapeutic response, xenografts serially sectioned on five levels were analysed for the presence

of tumour based on histology and expression of Pax8 and CA125.

At the completion of therapy, xenograft diminution was observed in all treatment groups but tumour regression was significantly higher in mice treated with the combination of carboplatin and birinapant (Po0.05 or 0.01 one-way ANOVA, Fig 6b and Supplementary Data 1) No evidence of disease was present in majority of xenografts treated with this co-therapy (9 of 13) based on histology and Pax8 expression, and in the minority of cases where disease was detected it was confined

to small foci (Supplementary Fig 8b and Supplementary Table 6) In contrast, significantly larger xenografts containing residual tumour were observed with carboplatin or birinapant monotherapy (Po0.05 or 0.01 one-way ANOVA, Fig 6b, Supplementary Data 1, Supplementary Fig 8b,c and Supplementary Table 6) Predominance of residual tumour cells were CA125 negative after carboplatin treatment (Supplementary Fig 8b) verifying therapy resistance in CA125-negative tumour cells Progression-free survival is an accepted metric for evaluation of ovarian cancer therapy47 In these experiments, therapeutic response to carboplatin and birinapant co-treatment was durable with no tumour progression (determined by response evaluation criteria in solid tumours (RECIST)) 1 month post therapy (Fig 6c and Supplementary Data 1) With co-therapy, tumour was undetectable in 6/11 xenografts and only small tumour foci were found in remaining xenografts (Fig 6c, Supplementary Data 1, Supplementary Fig 8d–f, and Supplementary Table 6) Tumour progression was observed in all monotherapy-treated mice (Fig 6c, Supplementary Data 1, Supplementary Fig 8c–f, Supplementary Data 2 and Supplementary Table 6), even in S5-GODL tumours that harbour a BRCA2 mutation48 Emergence of CA125-positive tumour cells was noted in all carboplatin-treated xenografts after therapy was stopped, demonstrating in vivo differentiation of the CA125-negative population after cessation of treatment (Supplementary Fig 8d) The biologic behaviour of control cell lines was as expected (Supplementary Fig 9a–c and Supplementary Data 2).

Similar results were obtained in repeat experiments where each mouse carried one xenograft generated from subcutaneous implantation of S3- or S5-GODL cells (Supplementary Fig 10)

or intraperitoneal injection of S1-GODL cells (Fig 7a) Tumour-bearing mice were treated for 4 weeks in these experiments Tumour elimination was observed in five of six subcutaneous xenografts immediately after therapy with no disease progression

6 weeks after cessation of birinapant and carboplatin co-therapy (Supplementary Fig 10a,b, Supplementary Data 2 and Supplementary Table 6) In the IP model, no tumour was found

at the completion of therapy in majority of birinapant and carboplatin-treated animals (three of four; Fig 7b,c and

Supplementary Table 6) Six weeks after cessation of therapy one

mouse remained disease free, while the remaining mice had only

microscopic traces of disease with no obvious tumour progression

(Fig 7b,c and Supplementary Table 6) Simultaneously, the serum

of these experimental mice was analysed for the presence and levels of human CA125 While a correlation between tumour

Carboplatin Birinapant

QNZ

– – – –

+ – – –

+ – + –

+ – – +

+ + – –

+ + + –

+ + – +

– – – –

+ – – –

+ – + –

+ – – +

+ + – –

+ + + –

+ + – +

0 20 40 60 80 100

B+C B+C +

0 20 40 60 80 100

αTNF

C + QNZ B+C B+C + αTNF B+C + QNZ

Annexin V

Annexin V

Annexin V

Annexin V

Annexin V

Annexin V

SSC

SSC

SSC

SSC

SSC

SSC

100%

0%

50%

50%

100%

50%

100%

0%

42.9%

57.1%

Carboplatin

Therapy

resistant

CA125– cells

Differentiated CA125+ cells

HGSC 1

Therapy resistant CA125– cells

Differentiated CA125+ cells

Therapy resistant CA125– cells

Differentiated CA125+ cells HGSC 2

HGSC 3

PARP

Erk

Cleaved PARP

pan-cIAP

XIAP

cIAP

Pro-caspase8 Caspase8

PARP Cleaved PARP

ERK

120 kDa

50 kDa

80 kDa

50 kDa

50 kDa

50 kDa

38 kDa

38 kDa

38 kDa

120 kDa

Vehicle Birinaapant Carboplatin Vehicle Birinapant Carboplatin Birinapant+

vehicle Birinapant Carboplatin carboplatin Vehicle Birinapant Carboplatin Birinapant+

Vehicle Birinapant Carboplatin carboplatin

50 kDa

50 kDa

50 kDa

CA125 – cells

CA125+

cells

C: Carboplatin B+C : Birinapant + carboplatin

CA125-negative cells (n =3)

CA125-positive cells (n =3)

Birinapant + carboplatin

99.8% death

99.9% death 99.9% death

0.2% death

Figure 5 | Birinapant mediated degradation of cIAP re-enabled apoptosis in the CA125-negative platinum-resistant cells (a) FACS analysis of CA125-negative and -positive cell populations isolated from primary chemo-naive HGSC and treated with drug revealed AnnexinV and propidium iodide positive apoptotic cell death in CA125-negative cells only after combined treatment with birinapant and carboplatin (n¼ 3 run in triplicate, representative sample shown) (b) Western blot of isolated tumour subpopulations from three independent HGSCs confirmed birinapant mediated activation of apoptosis based on degradation of cIAP proteins and presence of cleaved PARP, and decreased NF-kB signalling measured by reduction in phospho NF-kB (c) Birinapant-induced cell death in HGSC is TNFa dependent and is not abolished with inhibition of NF-kB signalling Isolated CA125-negative and -positive human HGSC populations were treated with carboplatin with or without birinapant and co-treated with either vehicle, anti-TNFa neutralizing antibody or

triplicate) Western blots were performed on cells isolated from these assays (n¼ 3, one representative sample is shown) Treatment of CA125-negative cells with birinapant and carboplatin caused degradation of cIAP, activated apoptosis as evidenced by cleavage of pro-caspase 8 to caspase 8 and its downstream target PARP, and resulted in cell death Therapeutic effects of birinapant in combination with carboplatin were TNFa dependent, evidenced by blockade of the apoptotic pathway (western blot) and cell death (graph) with anti-TNFa neutralizing antibody Blockade of NF-kB signalling by QNZ, evidenced by reduced NF-kB phosphorylation (western blot), was not sufficient to sensitize CA125-negative cells to carboplatin monotherapy nor could it block the birinapant and carboplatin co-therapy induced cell death (graphs) In all cases, CA125-positive cells were eliminated by treatment with carboplatin regardless of co-treatment with birinapant, anti-TNFa or QNZ

burden and serum CA125 levels was found in vehicle-treated

mice, carboplatin-treated animals had undetectable levels of

human serum CA125 immediately post therapy despite presence

of residual tumour (Fig 7d, Supplementary Data 1,

Supplementary Fig 10c and Supplementary Data 2)

Off-treatment CA125 levels rose in carboplatin-treated mice

(Fig 7d, Supplementary Data 1, Supplementary Fig 10c and

Supplementary Data 2), suggesting that serum CA125 may not

detect residual therapy-resistant CA125-negative cells.

Results of these pilot experiments imply that levels of the CA125 biomarker may only rise after the CA125-negative population containing cancer stem cells has had time to differentiate and give rise to CA125-positive progeny Further characterization of the CA125-negative population with respect

to their secretome may help elucidate more reliable biomarkers for detection of recurrent and perhaps early stage disease Our results demonstrated clear differences between the transcriptomes

of CA125-negative and -positive populations with respect to cell

(3 weeks)

Harvest Timecourse

Carboplatin

Birinapant

Harvest

Experimental cohorts:

Vehicle (n =8)

Birinapant (n =8)

Carboplatin (n =8)

Birinapant + carboplatin (n =8)

Release (4 weeks)

Response of tumors to therapy in vivo

0 1 10 100

0 1 10 100

0 1 10 100 1,000

0 1 10 100 1000 10,000

Vehicle BirinCarbo

Birin + carbo Vehicle BirinCarbo

Birin + carbo

Vehicle Birin Carbo

Birin + carbo

Vehicle Birin Carbo

Birin + carbo

Vehicle Birin Carbo

Birin + carbo

Vehicle Birin Carbo

Birin + carbo

0 1 10 100

0 1 10 100

0 20 40 60 80 100

Days after cessation of therapy

Vehicle Birinapant Carboplatin Birinapant + carboplatin

0 20 40 60 80 100

Days after cessation of therapy

Vehicle Birinapant Carboplatin Birinapant + carboplatin 0

20 40 60 80 100

Days after cessation of therapy

Vehicle Birinapant Carboplatin Birinapant + carboplatin

0 20 40 60 80

Days

Vehicle Birinapant Carboplatin Birinapant + carboplatin

0 10 20 30

Vehicle Birinapant Carboplatin Birinapant + carboplatin

Days 0

10 20 30

Vehicle Birinapant Carboplatin Birinapant + carboplatin

Days

Analysis of progression free survival

P < 0.05

P < 0.01

3 )

3 )

3 )

Figure 6 | Co-administration of birinapant with carboplatin led to decreased tumour burden and increased disease-free survival in vivo (a) Mice harbouring tumours from low passaged patient-derived HGSC cell lines were treated with vehicle (Vehicle, V), carboplatin (Carbo, C), birinapant (Birin, B)

or both drugs (Birinþ carbo, B þ C) for 3 weeks Xenografts were harvested from half the mice at completion of therapy (n ¼ 5 S1-GODL, n ¼ 4 S3- and

versus Bþ C ¼ 0.001, 0.009 and 0.005 for S1-, S3- and S5-GODL, respectively by unpaired two-sided t-test) (c) One month after cessation of therapy all mice in the co-therapy arm were progression free, in contrast to clear evidence of disease progression in all other treatment arms based on serial

unpaired two-sided t-test) Results are mean±s.e.m for tumour volume and median±IQR for tumour mass

surface antigenic profile and candidate-secreted factors

(Supplementary Tables 7 and 8) This data lays the groundwork

for further purifying HGSC tumour-initiating cells from the

CA125-negative subpopulation using positive selection markers

in future work.

We next examined the effects of in vivo birinapant and carboplatin co-treatment on xenografts raised directly from two platinum-sensitive HGSCs with opposite birinapant sensitivities (determined by in vitro assays) To further test whether the combinatorial therapeutic approach could also apply to HGSCs

Organs

Carboplatin

Birinapant + carboplatin HE

HE

Tumor

Tumor

Tumor

Tumor

Tumor

Six weeks after cessation of therapy

Carboplatin

Birinapant + carboplatin HE

HE

Tumor

No tumor

Tumor

Tumor

Vehicle

Carboplatin

Birinapant + carboplatin

Birinapant

Carboplatin

weekly x4 Birinapant

weekly x8

Initiate

intraperitoneal

tumors

2 weeks

Treat

4 weeks

Harvest response to therapy

Harvest re-growth after therapy

Release

6 weeks

S1-GODL

injected IP

Immediately post-therapy

0 400 800 1,200 1,600

Vehicle immediately post-therapy Pre-therapy

Vehicle 6 weeks off therapy

0 10 20 30 40

Carboplatin immediately post-therapy Pre-therapy

Carboplatin 6 weeks off therapy

Carboplatin

Birinapant + carboplatin

HE HE

Tumor cells

Tumor cells

Tumor cells

Tumor cells

Six weeks after cessation of therapy Immediately post-therapy

Carboplatin

Birinapant + carboplatin

HE HE

No tumor

Tumor cells

Tumor cells

Tumor cells

Pelvic wash cell pellets

Figure 7 | Birinapant and carboplatin co-therapy was efficacious against intraperitoneal tumours (a) Mice bearing intraperitoneal S1-GODL tumours were treated with vehicle, birinapant, carboplatin or birinapant and carboplatin for 4 weeks (n¼ 8 per group) Half the mice were sacrificed at the completion of therapy, and half 6 weeks after cessation of treatment (b) Immediately post therapy, organs of co-therapy treated mice were free of tumour in all except one (of four) where only small foci of tumours were detected Six weeks after cessation of therapy, two of four co-therapy treated mice had no evidence of tumour implants, while remaining two mice had only microscopic evidence of disease In contrast, multiple implanted tumours were

collected, enumerated and concentrated into a cell pellet for each mouse Immediately post therapy no tumour cells were detected in cell pellets from mice treated with birinapant and carboplatin co-therapy (n¼ 4) In contrast, tumour was detected in pelvic washings of all vehicle or birinapant-treated mice and

in one mouse treated with carboplatin (n¼ 4 per group) Six weeks after cessation of therapy, only minute traces of tumour cells were detected in pelvic wash pellets from three of four co-therapy treated mice Significant tumour burden was detected in all other cohorts Insets of CA125-stained tumour cells are shown for each cell pellet (n¼ 4 per group) Residual tumour was CA125 negative immediately following carboplatin therapy Scale bars are

500 mm in hemotoxylin and eosin images, and 100 mm in CA125 insets (d) Measurement of serum human CA125 in experimental mice demonstrated a correlation between tumour burden of S1-GODL cells and serum human CA125 in vehicle-treated mice No human serum CA125 could be detected in carboplatin-treated mice immediately post therapy In contrast, human CA125 was detected in serum of these mice 6 weeks after cessation of treatment

R2calculated by linear regression

Figure 8 | Response of patient-derived xenografts to co-therapy correlated with in vitro birinapant sensitivity (a) Carboplatin treatment predominantly eliminated differentiated CA125-positive cells but spared the CA125-negative population in two independent platinum-resistant HGSCs (n¼ 3 replicates) (b) Cells from a platinum-resistant HGSC with high cIAP levels in the CA125-negative population were eliminated by in vitro co-treatment with birinapant and carboplatin Birinapant therapy had minimal effect on the specimen with low cIAP expression regardless of carboplatin administration Results are mean±s.d.,

n¼ 3 replicates per sample Western blot analysis of cIAP levels in CA125-negative and -positive populations is shown (c) A significant decrease in tumour mass was observed in response to combination therapy only in human HGSCs that were Birinapant sensitive (patients I and III), regardless of sensitivity to platinum as a monotherapy Results are mean±s.e.m (n¼ 3 per group or n ¼ 5 for co-therapy treated patients I, II and IV], P value by unpaired two-sided t-test) (d) Immediately after therapy, residual tumour was detected in xenografts treated with carboplatin monotherapy regardless of clinical classification as platinum sensitive or resistant In the birinapant and platinum-sensitive sample (patient I), two of five xenografts were tumour free following co-therapy, with minimal disease in remaining xengrafts Similarly, in a chemo-resistant but birinapant-sensitive human HGSC (patient III) complete tumour resolution was observed in 2 of 3 xenografts Tumour was detected in all co-therapy treated xenografts generated from the birinapant insensitive samples (n¼ 5) Scale bars are 50 mm (e) CA125-negative residual tumour was detected in all patient-derived xenografts immediately after carboplatin monotherapy Scale bars equal

50 mm (f) Tumour-bearing mice (n¼ 3 per group) were released off-treatment for 4 weeks and during this time tumour volume was serially measured daily and plotted Mice that had harboured birinapant sensitive patient-derived xenografts (patient I and III) were disease free 1 month after the cessation of co-therapy based on RECIST criteria Disease progression was observed in all other treatment groups